Chromosomal assessment to diagnose urogenital malignancy in dogs

ABSTRACT

This invention is directed to a method of diagnosing bladder cancer in dogs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a § 371 U.S. National Stage of InternationalApplication PCT/US2014/65773, filed Nov. 14, 2014, which claims thebenefit of U.S. Provisional Application 61/904,659 filed Nov. 15, 2013,which are hereby incorporated by reference in their entireties.

1. FIELD OF THE INVENTION

This invention relates generally to the discovery of an improved methodto diagnose urogenital malignancy in dogs.

2. BACKGROUND OF THE INVENTION

2.1. Introduction

Transitional cell carcinoma (TCC), also referred to as urothelialcarcinoma (UC), is the most common urinary tract neoplasm in the dog.This form of cancer may be localized in one or more anatomical sites,including the kidneys, ureters, urinary bladder, prostate, and urethra,with most cases being detected in the bladder [1]. In the bladder, thecancer develops from the transitional epithelial cells that form thelining of the bladder and invade into the bladder wall and layers ofmuscle. As the mass becomes larger a frequent consequence is obstructionto the flow of urine, either from the kidneys to the bladder, or fromthe bladder through the urethra. Pathology assessment of canine TCCsindicates that most are higher grade tumors that have the ability tospread to lymph nodes and other organs in the body (lung, liver,others).

The American Veterinary Medical Association estimates that 4.2 milliondogs are diagnosed with cancer each year in the US. While preciselifetime risk and incidence numbers for TCC in the overall pet dogpopulation is not known, TCC is estimated to represent ˜1-2% of alldiagnosed cancers, indicating that as many as 40,000-80,000 dogs eachyear could develop TCC in the US. In addition there are several breedsof purebred dog that have been reported to have an elevated risk ofdeveloping TCC of the bladder, including Scottish terrier, Shetlandsheepdog, West Highland white terrier, Wire/Fox terrier and beagle.

2.2. The Diagnostic Challenge of Canine TCC

A major challenge to diagnosis of TCC in a dog is that the symptoms ofurothelial cancer in the dog are shared with variety of other urinarytract conditions. For example, bladder infections, bladder stones,hyperplastic growths in the bladder, and inflammation of the bladder canall cause symptoms in the dog similar to those resulting from a bladdercancer. Evaluation of canine urine by routine cytology may bemisleading, since the non-malignant conditions above may cause sheddingof abnormal looking cells in the urine, which may be mistaken for amalignancy. The use of imaging techniques, such as radiography andultrasonography, may identify the presence of unusual growths in theurinary tract, but these may or may not be malignant, and may alsoresult in the presence of abnormal looking cells in the urine.Presently, a confirmed diagnosis of a canine TCC may be made onlyfollowing the evaluation of a biopsy of the tumor by a pathologist.Obtaining a biopsy of a probable mass in the urinary tract may beperformed during surgery, cystoscopy or by traumatic catheterization,which have a decreasing level of intervention, respectively. However,any procedure that disturbs the likely tumor mass may result in seedingof malignant epithelial cells elsewhere on the local area, resulting inspreading of the cancer. The chances of ‘seeding’ are of concern to theclinical management. Confirmation of diagnosis of a TCC in dogspresenting with symptoms suggestive of a TCC would thus be desirablefrom an assessment of a free catch urine sample.

3. SUMMARY OF THE INVENTION

In particular non-limiting embodiments, the present invention provides amethod for detecting a urogenital malignancy in a biological sample froma dog which comprises: (a) measuring a copy number of CFA 13, CFA 19, orCFA 36; and (b) if the copy number of CFA 13 or CFA 36 is elevated, orCFA 19 is reduced, from that of a normal control, determining that thedog has increased likelihood of the urogenital malignancy. In oneembodiment, the copy number of CFA 13, CFA 19 and CFA 36 are measured.

The copy number may be measured by fluorescence in situ hybridization(FISH), polymerase chain reaction (PCR), comparative genomichybridization (CGH), or next generation sequencing. The biologicalsample may be a urine sample, a fresh-frozen sample, a fresh sample, ora formalin-fixed, paraffin-embedded sample.

The invention also provides a method of selecting a dog for urogenitalmalignancy treatment which comprises measuring a copy number of CFA 13,CFA 19, or CFA 36 in a biological sample from a dog; and if the copynumber of CFA 13 or CFA 36 is elevated, or CFA 19 is reduced, from thatof a normal control, selecting the dog for urogenital malignancytreatment. The urogenital malignancy treatment may be include surgery,radiation therapy or chemotherapy.

A method of diagnosing a urogenital malignancy in a sample from a dogcomprising: (a) detecting the copy number of CFA 13, CFA 19 or CFA 36 ina sample from the dog, by a nucleic acid hybridization assay withnucleic acids specific for CFA 13, CFA 19 or CFA 36; (b) comparing thedetected levels to at least one sample from a training set(s), wherein asample training set(s) comprises data from the levels from a referencesample, and the comparing step comprises applying a statisticalalgorithm which comprises determining a correlation between the detectedlevels in the sample from the subject and the detected levels from atleast one training set(s); and (c) diagnosing the urogenital malignancybased on the detected levels in the sample from the subject and theresults of the statistical algorithm. The nucleic acid hybridizationassay may be FISH analysis.

A method of diagnosing a urogenital malignancy in a sample from a dogcomprising: (a) detecting the copy number of CFA 13, CFA 19 or CFA 36 ina sample from the dog, by a digital droplet PCR assay withprimers/probes specific for CFA 13, CFA 19 or CFA 36; (b) comparing thedetected levels to at least one sample from a training set(s), wherein asample training set(s) comprises data from the levels from a referencesample, and the comparing step comprises applying a statisticalalgorithm which comprises determining a correlation between the detectedlevels in the sample from the subject and the detected levels from atleast one training set(s); and (c) diagnosing the urogenital malignancybased on the detected levels in the sample from the subject and theresults of the statistical algorithm.

In addition, the invention provides a kit for detecting a urogenitalmalignancy in a dog comprising: (a) at least one reagent selected fromthe group consisting of: a nucleic acid probe capable of specificallydetecting CFA 13, CFA 19 or CFA 36; and (b) instructions for use inmeasuring a copy number of CFA 13, CFA 19 or CFA 36 in a biologicalsample from a dog wherein if the copy number of CFA 13 or CFA 36 iselevated, or CFA 19 is reduced, from that of a normal control.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the significant copy number variations based on analysis of31 cases of canine TCC.

FIG. 2 shows multicolor FISH hybridization using probes designed todetect and quantify dog chromosomes 8, 13, 19 and 36.

FIG. 3 shows multicolor FISH hybridization using probes designed todetect and quantify dog chromosomes 8, 13, 19 and 36. Panel (A) is ahealthy dog and panels (B) and (C) are confirmed cases of TCC.

FIG. 4 shows the results of both FISH copy number analysis and analternative embodiment, PCR method, to measure copy number.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Definitions

“Urogenital malignancy” is cancer that forms in the tissues of thebladder or neighboring tissues. Urogenital malignancy is used herein toinclude transitional cell carcinoma (TCC), which is also referred to asurothelial carcinoma. The methods and reagents described herein may alsobe used to detect squamous cell carcinoma and adenocarcinoma.

“Copy number” is a measurement of DNA, whether of a single locus, one ormore loci, or an entire genome. A “copy number” of two is “wild-type” ina dog (because of diploidy, except for sex chromosomes). A “copy number”of other than two in a dog (except for sex chromosomes) deviates fromwild-type. Such deviations include gains and amplifications, i.e.,increases in copy numbers, and deletions, i.e., decreases in copynumbers and even the absence of copy numbers.

“Labeled,” “labeled with a detectable label,” and “detectably labeled”are used interchangeably herein to indicate that an entity (e.g., aprobe) can be detected. “Label” and “detectable label” mean a moietyattached to an entity to render the entity detectable, such as a moietyattached to a probe to render the probe detectable upon binding to atarget sequence. The moiety, itself, may not be detectable but maybecome detectable upon reaction with yet another moiety. Use of the term“detectably labeled” is intended to encompass such labeling.

The detectable label can be selected such that the label generates asignal, which can be measured and the intensity of which is proportionalto the amount of bound entity. A wide variety of systems for labelingand/or detecting molecules, such as nucleic acids, e.g., probes, arewell-known. Labeled nucleic acids can be prepared by incorporating orconjugating a label that is directly or indirectly detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical, chemical or other means. Suitable detectable labels includeradioisotopes, fluorophores, chromophores, chemiluminescent agents,microparticles, enzymes, magnetic particles, electron dense particles,mass labels, spin labels, haptens, and the like. Fluorophores andchemiluminescent agents are preferred herein.

“Nucleic acid sample” refers to a sample comprising nucleic acid in aform suitable for hybridization with a probe, such as a samplecomprising nuclei or nucleic acids isolated or purified from suchnuclei. The nucleic acid sample may comprise total or partial (e.g.,particular chromosome(s)) genomic DNA, total or partial mRNA (e.g.,particular chromosome(s) or gene(s)), or selected sequence(s). Condensedchromosomes (such as are present in interphase or metaphase) aresuitable for use as targets in in situ hybridization, such as FISH.

“Predetermined cutoff” and “predetermined level” refer generally to acutoff value that is used to assess diagnostic/prognostic/therapeuticefficacy results by comparing the assay results against thepredetermined cutoff/level, where the predetermined cutoff/level alreadyhas been linked or associated with various clinical parameters (e.g.,severity of disease, progression/nonprogression/improvement, etc.).

“Probe,” in the context of the present disclosure, is an oligonucleotideor polynucleotide that can selectively hybridize to at least a portionof a target sequence under conditions that allow for or promoteselective hybridization. In general, a probe can be complementary to thecoding or sense (+) strand of DNA or complementary to the non-coding oranti-sense (−) strand of DNA (sometimes referred to as“reverse-complementary”). Probes can vary significantly in length. Alength of about 10 to about 100 nucleotides, such as about 15 to about75 nucleotides, e.g., about 15 to about 50 nucleotides, can be preferredin some applications such as PCR, whereas a length of about 50 to about1×10⁶ nucleotides can be preferred for chromosomal probes and a lengthof about 5,000 to about 800,000 nucleotides or more preferably about100,000 to about 400,000 for BAC probes.

The invention encompasses fragments of a nucleic acids that can serve(1) as probes for detecting segments of domestic dog (Canis familairis,CFA) genome referred to as chromosomes 13, 19 or 36 (hereafter referredto as CFA 13, CFA 19 and CFA 36). The dog genome has been sequenced andis available for example, the NCBI Canis lupus familiaris genomedatabase; or ENSEMBL database CanFam3.1 (GCA_000002285.2). See also,Lindblad-Toh et al. 2005 “Genome sequence, comparative analysis andhaplotype structure of the domestic dog” Nature 438 (7069), 803-819.

The changes in CFA 13, 19 or 36 may be detected by a number of methodswell known in the art, e.g. Southern and northern blotting, dotblotting, colony hybridizations, hybridization to an array, comparativegenomic hybridization (CGH), etc. or (2) as polymerase chain reaction(PCR) primers to amplify CFA 13, 19 or 36. PCR primers can comprise, inaddition to CFA 13, 19 or 36 nucleic acid sequences, other sequencessuch as restriction enzyme cleavage sites that facilitate the use of theamplified nucleic acid. PCR is described in the following references:Saiki et al. 1988 Science 239 487-491; PCR Technology, Erlich, ed.,Stockton Press, (1989). As explained below, PCR can be useful to detectabnormally low or high levels of CFA 13, 19 or 36.

Hybridization techniques are well known in the art and are described bySambrook, J., E. F. Fritsch, and T. Maniatis (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11, (1989)) and Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4 (1995)), the relevant portions of which areincorporated by reference herein. Moderately stringent conditions forfilter hybridizations include hybridization in about 50% formamide,6×SSC at a temperature from about 42° C. to 55° C. and washing at about60° C. in 0.5×SSC, 0.1% SDS. Highly stringent conditions are defined ashybridization conditions as above, but with washing at approximately 68°C. in 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH₂PO₄, and1.26 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15 M NaCland 1 5 mM sodium citrate) in the hybridization and wash buffers;washes, optionally at least two washes, are performed for 15 minutesafter hybridization is complete.

It should be understood that the wash temperature and wash saltconcentration can be adjusted as necessary to achieve a desired degreeof stringency by applying the basic principles that govern hybridizationreactions and duplex stability, as known to those skilled in the art anddescribed further below (see e.g., Sambrook et al., supra). When nucleicacids of known sequence are hybridized, the hybrid length can bedetermined by aligning the sequences of the nucleic acids (for example,using GAP) and identifying the region or regions of optimal sequencecomplementarity. The hybridization temperature for hybrids anticipatedto be less than 50 base pairs in length should be 5 to 10° C. less thanthe melting temperature (Tm) of the hybrid, where Tm is determinedaccording to the following equations. For hybrids less than 18 basepairs in length, Tm (° C.)=2(# of A+T bases)+4(# of G+C bases). Forhybrids above 18 base pairs in length, Tm (°C.)=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)−(600 N), where N is the number ofbases in the hybrid, and [Na+] is the concentration of sodium ions inthe hybridization buffer. Each such hybridizing nucleic acid has alength that is at least 15 nucleotides (or at least 18 nucleotides, orat least 20, or at least 25, or at least 30, or at least 40, or at least50, or at least 100. Sambrook et al., supra.

5.2. Polynucleotide Amplification and Determination

In many instances, it is desirable to amplify a nucleic acid sequenceusing any of several nucleic acid amplification procedures which arewell known in the art. Specifically, nucleic acid amplification is thechemical or enzymatic synthesis of nucleic acid copies which contain asequence that is complementary to a nucleic acid sequence beingamplified (template). The methods and kits of the invention may use anynucleic acid amplification or detection methods known to one skilled inthe art, such as those described in U.S. Pat. No. 5,525,462 (Takarada etal.); U.S. Pat. No. 6,114,117 (Hepp et al.); U.S. Pat. No. 6,127,120(Graham et al.); U.S. Pat. No. 6,344,317 (Urnovitz); U.S. Pat. No.6,448,001 (Oku); U.S. Pat. No. 6,528,632 (Catanzariti et al.); and PCTPub. No. WO 2005/111209 (Nakajima et al.); all of which are incorporatedherein by reference in their entirety.

Commonly used methods known in the art for the quantification of mRNAexpression in a sample include northern blotting and in situhybridization (Parker and Barnes, Methods Mol. Biol. 106:247-83, 1999),RNAse protection assays (Hod, Biotechniques 13:852-54, 1992), PCR-basedmethods, such as reverse transcription PCR(RT-PCR) (Weis et al., TIG8:263-64, 1992), and array-based methods (Schena et al., Science270:467-70, 1995). Alternatively, antibodies may be employed that canrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes, or DNA-protein duplexes. Representative methodsfor sequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), bead-based technologies, single moleculefluorescence in situ hybridization (smFISH) studies, and gene expressionanalysis by massively parallel signature sequencing. Velculescu et al.1995 Science 270 484-487; Streefkerk et al., 1976, Pro Biol Fluid ProcColl 24 811-814; Soini U.S. Pat. No. 5,028,545; smFISH, Lyubimova et al.2013 Nat Protocol 8(9) 1743-1758.

In some embodiments, the nucleic acids are amplified by PCRamplification using methodologies known to one skilled in the art. Oneskilled in the art will recognize, however, that amplification can beaccomplished by any known method, such as ligase chain reaction (LCR),Qβ-replicase amplification, rolling circle amplification, transcriptionamplification, self-sustained sequence replication, nucleic acidsequence-based amplification (NASBA), each of which provides sufficientamplification. Branched-DNA technology may also be used to qualitativelydemonstrate the presence of a sequence of the technology, whichrepresents a particular methylation pattern, or to quantitativelydetermine the amount of this particular genomic sequence in a sample.Nolte reviews branched-DNA signal amplification for direct quantitationof nucleic acid sequences in clinical samples (Nolte, 1998, Adv. Clin.Chem. 33:201-235).

The PCR process is well known in the art and is thus not described indetail herein. For a review of PCR methods and protocols, see, e.g.,Innis et al., eds., PCR Protocols, A Guide to Methods and Application,Academic Press, Inc., San Diego, Calif. 1990; U.S. Pat. No. 4,683,202(Mullis); which are incorporated herein by reference in their entirety.PCR reagents and protocols are also available from commercial vendors,such as Roche Molecular Systems. PCR may be carried out as an automatedprocess with a thermostable enzyme. In this process, the temperature ofthe reaction mixture is cycled through a denaturing region, a primerannealing region, and an extension reaction region automatically.Machines specifically adapted for this purpose are commerciallyavailable.

5.3. High Throughput, Single Molecule Sequencing, and Direct DetectionTechnologies

Suitable next generation sequencing technologies are widely available.Examples include the 454 Life Sciences platform (Roche, Branford, Conn.)(Margulies et al. 2005 Nature, 437, 376-380); lllumina's GenomeAnalyzer, GoldenGate Methylation Assay, or Infinium Methylation Assays,i.e., Infinium HumanMethylation 27K BeadArray or VeraCode GoldenGatemethylation array (Illumina, San Diego, Calif.; Bibkova et al., 2006,Genome Res. 16, 383-393; U.S. Pat. Nos. 6,306,597 and 7,598,035(Macevicz); U.S. Pat. No. 7,232,656 (Balasubramanian et al.)); or DNASequencing by Ligation, SOLiD System (Applied Biosystems/LifeTechnologies; U.S. Pat. Nos. 6,797,470, 7,083,917, 7,166,434, 7,320,865,7,332,285, 7,364,858, and 7,429,453 (Barany et al.); or the Helicos TrueSingle Molecule DNA sequencing technology (Harris et al., 2008 Science,320, 106-109; U.S. Pat. Nos. 7,037,687 and 7,645,596 (Williams et al.);U.S. Pat. No. 7,169,560 (Lapidus et al.); U.S. Pat. No. 7,769,400(Harris)), the single molecule, real-time (SMRT™) technology of PacificBiosciences, and sequencing (Soni and Meller, 2007, Clin. Chem. 53,1996-2001) which are incorporated herein by reference in their entirety.These systems allow the sequencing of many nucleic acid moleculesisolated from a specimen at high orders of multiplexing in a parallelfashion (Dear, 2003, Brief Funct. Genomic Proteomic, 1(4), 397-416 andMcCaughan and Dear, 2010, J. Pathol., 220, 297-306). Each of theseplatforms allow sequencing of clonally expanded or non-amplified singlemolecules of nucleic acid fragments. Certain platforms involve, forexample, (i) sequencing by ligation of dye-modified probes (includingcyclic ligation and cleavage), (ii) pyrosequencing, and (iii)single-molecule sequencing.

Pyrosequencing is a nucleic acid sequencing method based on sequencingby synthesis, which relies on detection of a pyrophosphate released onnucleotide incorporation. Generally, sequencing by synthesis involvessynthesizing, one nucleotide at a time, a DNA strand complimentary tothe strand whose sequence is being sought. Study nucleic acids may beimmobilized to a solid support, hybridized with a sequencing primer,incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase,adenosine 5′ phosphsulfate and luciferin. Nucleotide solutions aresequentially added and removed. Correct incorporation of a nucleotidereleases a pyrophosphate, which interacts with ATP sulfurylase andproduces ATP in the presence of adenosine 5′ phosphsulfate, fueling theluciferin reaction, which produces a chemiluminescent signal allowingsequence determination. Machines for pyrosequencing and methylationspecific reagents are available from Qiagen, Inc. (Valencia, Calif.).See also Tost and Gut, 2007, Nat. Prot. 2 2265-2275. An example of asystem that can be used by a person of ordinary skill based onpyrosequencing generally involves the following steps: ligating anadaptor nucleic acid to a study nucleic acid and hybridizing the studynucleic acid to a bead; amplifying a nucleotide sequence in the studynucleic acid in an emulsion; sorting beads using a picoliter multiwellsolid support; and sequencing amplified nucleotide sequences bypyrosequencing methodology (e.g., Nakano et al., 2003, J. Biotech. 102,117-124). Such a system can be used to exponentially amplifyamplification products generated by a process described herein, e.g., byligating a heterologous nucleic acid to the first amplification productgenerated by a process described herein.

Certain single-molecule sequencing embodiments are based on theprincipal of sequencing by synthesis, and utilize single-pairFluorescence Resonance Energy Transfer (single pair FRET) as a mechanismby which photons are emitted as a result of successful nucleotideincorporation. The emitted photons often are detected using intensifiedor high sensitivity cooled charge-couple-devices in conjunction withtotal internal reflection microscopy (TIRM). Photons are only emittedwhen the introduced reaction solution contains the correct nucleotidefor incorporation into the growing nucleic acid chain that issynthesized as a result of the sequencing process. In FRET basedsingle-molecule sequencing or detection, energy is transferred betweentwo fluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5,through long-range dipole interactions. The donor is excited at itsspecific excitation wavelength and the excited state energy istransferred, non-radiatively to the acceptor dye, which in turn becomesexcited. The acceptor dye eventually returns to the ground state byradiative emission of a photon. The two dyes used in the energy transferprocess represent the “single pair”, in single pair FRET. Cy3 often isused as the donor fluorophore and often is incorporated as the firstlabeled nucleotide. Cy5 often is used as the acceptor fluorophore and isused as the nucleotide label for successive nucleotide additions afterincorporation of a first Cy3 labeled nucleotide. The fluorophoresgenerally are within 10 nanometers of each other for energy transfer tooccur successfully. Bailey et al. recently reported a highly sensitive(15 pg methylated DNA) method using quantum dots to detect methylationstatus using fluorescence resonance energy transfer (MS-qFRET) (Baileyet al. 2009, Genome Res. 19(8), 1455-1461, which is incorporated hereinby reference in its entirety).

An example of a system that can be used based on single-moleculesequencing generally involves hybridizing a primer to a study nucleicacid to generate a complex; associating the complex with a solid phase;iteratively extending the primer by a nucleotide tagged with afluorescent molecule; and capturing an image of fluorescence resonanceenergy transfer signals after each iteration (e.g., Braslaysky et al.,PNAS 100(7): 3960-3964 (2003); U.S. Pat. No. 7,297,518 (Quake et al.)which are incorporated herein by reference in their entirety). Such asystem can be used to directly sequence amplification products generatedby processes described herein. In some embodiments the released linearamplification product can be hybridized to a primer that containssequences complementary to immobilized capture sequences present on asolid support, a bead or glass slide for example. Hybridization of theprimer-released linear amplification product complexes with theimmobilized capture sequences, immobilizes released linear amplificationproducts to solid supports for single pair FRET based sequencing bysynthesis. The primer often is fluorescent, so that an initial referenceimage of the surface of the slide with immobilized nucleic acids can begenerated. The initial reference image is useful for determininglocations at which true nucleotide incorporation is occurring.Fluorescence signals detected in array locations not initiallyidentified in the “primer only” reference image are discarded asnon-specific fluorescence. Following immobilization of theprimer-released linear amplification product complexes, the boundnucleic acids often are sequenced in parallel by the iterative steps of,a) polymerase extension in the presence of one fluorescently labelednucleotide, b) detection of fluorescence using appropriate microscopy,TIRM for example, c) removal of fluorescent nucleotide, and d) return tostep a with a different fluorescently labeled nucleotide.

The technology may be practiced with digital PCR. Digital PCR wasdeveloped by Kalinina and colleagues (Kalinina et al., 1997, NucleicAcids Res. 25; 1999-2004) and further developed by Vogelstein andKinzler (1999, Proc. Natl. Acad. Sci. U.S.A. 96; 9236-9241). Theapplication of digital PCR is described by Cantor et al. (PCT Pub. Nos.WO 2005/023091A2 (Cantor et al.); WO 2007/092473 A2, (Quake et al.)),which are hereby incorporated by reference in their entirety. DigitalPCR takes advantage of nucleic acid (DNA, cDNA or RNA) amplification ona single molecule level, and offers a highly sensitive method forquantifying low copy number nucleic acid. Fluidigm® Corporation,BioRad's Digital PCR and Raindance technologies all offer systems forthe digital analysis of nucleic acids. See, Karlin-Neumann G et al.(2012). Probing copy number variations using Bio-Rad's QX100/200™Droplet Digital™ PCR system. Bio-Rad Bulletin 6277; Diderot et al.,Clinical Chemistry February 2013 clinchem.2012.193409.

In some embodiments, nucleotide sequencing may be by solid phase singlenucleotide sequencing methods and processes. Solid phase singlenucleotide sequencing methods involve contacting sample nucleic acid andsolid support under conditions in which a single molecule of samplenucleic acid hybridizes to a single molecule of a solid support. Suchconditions can include providing the solid support molecules and asingle molecule of sample nucleic acid in a “microreactor.” Suchconditions also can include providing a mixture in which the samplenucleic acid molecule can hybridize to solid phase nucleic acid on thesolid support. Single nucleotide sequencing methods useful in theembodiments described herein are described in PCT Pub. No. WO2009/091934 (Cantor).

In certain embodiments, nanopore sequencing detection methods include(a) contacting a nucleic acid for sequencing (“base nucleic acid,” e.g.,linked probe molecule) with sequence-specific detectors, underconditions in which the detectors specifically hybridize tosubstantially complementary subsequences of the base nucleic acid; (b)detecting signals from the detectors and (c) determining the sequence ofthe base nucleic acid according to the signals detected. In certainembodiments, the detectors hybridized to the base nucleic acid aredisassociated from the base nucleic acid (e.g., sequentiallydissociated) when the detectors interfere with a nanopore structure asthe base nucleic acid passes through a pore, and the detectorsdisassociated from the base sequence are detected.

A detector also may include one or more regions of nucleotides that donot hybridize to the base nucleic acid. In some embodiments, a detectoris a molecular beacon. A detector often comprises one or more detectablelabels independently selected from those described herein. Eachdetectable label can be detected by any convenient detection processcapable of detecting a signal generated by each label (e.g., magnetic,electric, chemical, optical and the like). For example, a CD camera canbe used to detect signals from one or more distinguishable quantum dotslinked to a detector.

Next generation sequencing techniques may be applied to measureexpression levels or count numbers of transcripts using RNA-seq or wholetranscriptome shotgun sequencing. See, e.g., Mortazavi et al. 2008 NatMeth 5(7) 621-627 or Wang et al. 2009 Nat Rev Genet 10(1) 57-63.

Nucleic acids in the invention may be counted using methods known in theart. In one embodiment, NanoString's n Counter system may be used. Geisset al. 2008 Nat Biotech 26(3) 317-325; U.S. Pat. No. 7,473,767(Dimitrov). Alternatively, Fluidigm's Dynamic Array system may be used.Byrne et al. 2009 PLoS ONE 4 e7118; Helzer et al. 2009 Can Res 697860-7866. For reviews, see also Zhao et al. 2011 Sci China Chem 54(8)1185-1201 and Ozsolak and Milos 2011 Nat Rev Genet 12 87-98.

The invention encompasses any method known in the art for enhancing thesensitivity of the detectable signal in such assays, including, but notlimited to, the use of cyclic probe technology (Bakkaoui et al., 1996,BioTechniques 20: 240-8, which is incorporated herein by reference inits entirety); and the use of branched probes (Urdea et al., 1993, Clin.Chem. 39, 725-6; which is incorporated herein by reference in itsentirety). The hybridization complexes are detected according towell-known techniques in the art.

Reverse transcribed or amplified nucleic acids may be modified nucleicacids. Modified nucleic acids can include nucleotide analogs, and incertain embodiments include a detectable label and/or a capture agent.Examples of detectable labels include, without limitation, fluorophores,radioisotopes, colorimetric agents, light emitting agents,chemiluminescent agents, light scattering agents, enzymes and the like.Examples of capture agents include, without limitation, an agent from abinding pair selected from antibody/antigen, antibody/antibody,antibody/antibody fragment, antibody/antibody receptor, antibody/proteinA or protein G, hapten/anti-hapten, biotin/avidin, biotin/streptavidin,folic acid/folate binding protein, vitamin B12/intrinsic factor,chemical reactive group/complementary chemical reactive group (e.g.,sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides) pairs, and the like. Modified nucleic acids having a captureagent can be immobilized to a solid support in certain embodiments.

The invention described herein may be used in conjunction with othermolecular techniques for detection of cancer such as US Pat Pub2013/0171637 (Giafis et al.) the contents of which are herebyincorporated by reference in its entirety.

5.4. Statistical Methods

The data may be ranked for its ability to distinguish biomarkers in boththe 1 versus all (i.e., disease versus normal) and the all-pairwise(i.e., normal versus specific disease) cases. One statistic used for theranking is the area under the receiver operator characteristic (ROC)curve (a plot of sensitivity versus (1-specificity)). Althoughbiomarkers are evaluated for reliability across datasets, theindependent sample sets are not combined for the purposes of the ROCranking. As a result, multiple independent analyses are performed andmultiple independent rankings are obtained for each biomarker's abilityto distinguish groups of interest.

It is to be understood that other genes and/or diagnostic criteria maybe used in this invention. For example, animal characteristics, standardblood workups, the results of imaging tests, and/or histologicalevaluation may optionally be combined with biomarkers disclosed herein.

Such analysis methods may be used to form a predictive model, and thenuse that model to classify test data. For example, one convenient andparticularly effective method of classification employs multivariatestatistical analysis modeling, first to form a model (a “predictivemathematical model”) using data (“modeling data”) from samples of knownclass (e.g., from subjects known to have, or not have, a particularclass, subclass or grade of lung cancer), and second to classify anunknown sample (e.g., “test data”), according to lung cancer status.

Pattern recognition (PR) methods have been used widely to characterizemany different types of problems ranging for example over linguistics,fingerprinting, chemistry and psychology. In the context of the methodsdescribed herein, pattern recognition is the use of multivariatestatistics, both parametric and non-parametric, to analyze spectroscopicdata, and hence to classify samples and to predict the value of somedependent variable based on a range of observed measurements. There aretwo main approaches. One set of methods is termed “unsupervised” andthese simply reduce data complexity in a rational way and also producedisplay plots which can be interpreted by the human eye. The otherapproach is termed “supervised” whereby a training set of samples withknown class or outcome is used to produce a mathematical model and isthen evaluated with independent validation data sets.

Unsupervised PR methods are used to analyze data without reference toany other independent knowledge. Examples of unsupervised patternrecognition methods include principal component analysis (PCA),hierarchical cluster analysis (HCA), and non-linear mapping (NLM).

Alternatively, and in order to develop automatic classification methods,it has proved efficient to use a “supervised” approach to data analysis.Here, a “training set” of biomarker expression data is used to constructa statistical model that predicts correctly the “class” of each sample.This training set is then tested with independent data (referred to as atest or validation set) to determine the robustness of thecomputer-based model. These models are sometimes termed “expertsystems,” but may be based on a range of different mathematicalprocedures. Supervised methods can use a data set with reduceddimensionality (for example, the first few principal components), buttypically use unreduced data, with all dimensionality. In all cases themethods allow the quantitative description of the multivariateboundaries that characterize and separate each class, for example, eachclass of lung cancer in terms of its biomarker expression profile. It isalso possible to obtain confidence limits on any predictions, forexample, a level of probability to be placed on the goodness of fit(see, for example, Sharaf; Illman; Kowalski, eds. (1986). Chemometrics.New York: Wiley). The robustness of the predictive models can also bechecked using cross-validation, by leaving out selected samples from theanalysis.

Examples of supervised pattern recognition methods include the followingnearest centroid methods (Dabney 2005 Bioinformatics 21(22):4148-4154and Tibshirani et al. 2002 Proc. Natl. Acad. Sci. USA 99(10):6576-6572);soft independent modeling of class analysis (SIMCA) (see, for example,Wold, (1977) Chemometrics: theory and application 52: 243-282.); partialleast squares analysis (PLS) (see, for example, Wold (1966) Multivariateanalysis 1: 391-420; Joreskog (1982) Causality, structure, prediction 1:263-270); linear discriminant analysis (LDA) (see, for example, Nillson(1965). Learning machines. New York.); K-nearest neighbor analysis (KNN)(see, for example, Brown and Martin 1996 J Chem Info Computer Sci36(3):572-584); artificial neural networks (ANN) (see, for example,Wasserman (1993). Advanced methods in neural computing. John Wiley &Sons, Inc; O'Hare & Jennings (Eds.). (1996). Foundations of distributedartificial intelligence (Vol. 9). Wiley); probabilistic neural networks(PNNs) (see, for example, Bishop & Nasrabadi (2006). Pattern recognitionand machine learning (Vol. 1, p. 740). New York: Springer; Specht,(1990). Probabilistic neural networks. Neural networks, 3(1), 109-118);rule induction (RI) (see, for example, Quinlan (1986) Machine learning,1(1), 81-106); and, Bayesian methods (see, for example, Bretthorst(1990). An introduction to parameter estimation using Bayesianprobability theory. In Maximum entropy and Bayesian methods (pp. 53-79).Springer Netherlands; Bretthorst, G. L. (1988). Bayesian spectrumanalysis and parameter estimation (Vol. 48). New York: Springer-Verlag);unsupervised hierarchical clustering (see for example Herrero 2001Bioinformatics 17(2) 126-136). In one embodiment, the classifier is thecentroid based method described in Mullins et al. 2007 Clin Chem53(7):1273-9, which is herein incorporated by reference in its entiretyfor its teachings regarding disease classification.

It is often useful to pre-process data, for example, by addressingmissing data, translation, scaling, weighting, etc. Multivariateprojection methods, such as principal component analysis (PCA) andpartial least squares analysis (PLS), are so-called scaling sensitivemethods. By using prior knowledge and experience about the type of datastudied, the quality of the data prior to multivariate modeling can beenhanced by scaling and/or weighting. Adequate scaling and/or weightingcan reveal important and interesting variation hidden within the data,and therefore make subsequent multivariate modeling more efficient.Scaling and weighting may be used to place the data in the correctmetric, based on knowledge and experience of the studied system, andtherefore reveal patterns already inherently present in the data.

If possible, missing data, for example gaps in column values, should beavoided. However, if necessary, such missing data may replaced or“filled” with, for example, the mean value of a column (“mean fill”); arandom value (“random fill”); or a value based on a principal componentanalysis (“principal component fill”). Each of these differentapproaches will have a different effect on subsequent PR analysis.

“Translation” of the descriptor coordinate axes can be useful. Examplesof such translation include normalization and mean centering.“Normalization” may be used to remove sample-to-sample variation. Manynormalization approaches are possible, and they can often be applied atany of several points in the analysis. “Mean centering” may be used tosimplify interpretation. Usually, for each descriptor, the average valueof that descriptor for all samples is subtracted. In this way, the meanof a descriptor coincides with the origin, and all descriptors are“centered” at zero. In “unit variance scaling,” data can be scaled toequal variance. Usually, the value of each descriptor is scaled by1/StDev, where StDev is the standard deviation for that descriptor forall samples. “Pareto scaling” is, in some sense, intermediate betweenmean centering and unit variance scaling. In pareto scaling, the valueof each descriptor is scaled by 1/sqrt(StDev), where StDev is thestandard deviation for that descriptor for all samples. In this way,each descriptor has a variance numerically equal to its initial standarddeviation. The pareto scaling may be performed, for example, on raw dataor mean centered data.

“Logarithmic scaling” may be used to assist interpretation when datahave a positive skew and/or when data spans a large range, e.g., severalorders of magnitude. Usually, for each descriptor, the value is replacedby the logarithm of that value. In “equal range scaling,” eachdescriptor is divided by the range of that descriptor for all samples.In this way, all descriptors have the same range, that is, 1. However,this method is sensitive to presence of outlier points. In“autoscaling,” each data vector is mean centred and unit variancescaled. This technique is a very useful because each descriptor is thenweighted equally and large and small values are treated with equalemphasis. This can be important for analytes present at very low, butstill detectable, levels.

Several supervised methods of scaling data are also known. Some of thesecan provide a measure of the ability of a parameter (e.g., a descriptor)to discriminate between classes, and can be used to improveclassification by stretching a separation. For example, in “varianceweighting,” the variance weight of a single parameter (e.g., adescriptor) is calculated as the ratio of the inter-class variances tothe sum of the intra-class variances. A large value means that thisvariable is discriminating between the classes. For example, if thesamples are known to fall into two classes (e.g., a training set), it ispossible to examine the mean and variance of each descriptor. If adescriptor has very different mean values and a small variance, then itwill be good at separating the classes. “Feature weighting” is a moregeneral description of variance weighting, where not only the mean andstandard deviation of each descriptor is calculated, but otherwell-known weighting factors, such as the Fisher weight, are used.

The methods described herein may be implemented and/or the resultsrecorded using any device capable of implementing the methods and/orrecording the results. Examples of devices that may be used include butare not limited to electronic computational devices, including computersof all types. When the methods described herein are implemented and/orrecorded in a computer, the computer program that may be used toconfigure the computer to carry out the steps of the methods may becontained in any computer readable medium capable of containing thecomputer program. Examples of computer readable medium that may be usedinclude but are not limited to diskettes, CD-ROMs, DVDs, ROM, RAM, andother memory and computer storage devices. The computer program that maybe used to configure the computer to carry out the steps of the methodsand/or record the results may also be provided over an electronicnetwork, for example, over the internet, an intranet, or other network.

The process of comparing a measured value and a reference value can becarried out in any convenient manner appropriate to the type of measuredvalue and reference value for the discriminative gene at issue.“Measuring” can be performed using quantitative or qualitativemeasurement techniques, and the mode of comparing a measured value and areference value can vary depending on the measurement technologyemployed. For example, when a qualitative colorimetric assay is used tomeasure expression levels, the levels may be compared by visuallycomparing the intensity of the colored reaction product, or by comparingdata from densitometric or spectrometric measurements of the coloredreaction product (e.g., comparing numerical data or graphical data, suchas bar charts, derived from the measuring device). However, it isexpected that the measured values used in the methods of the inventionwill most commonly be quantitative values. In other examples, measuredvalues are qualitative. As with qualitative measurements, the comparisoncan be made by inspecting the numerical data, or by inspectingrepresentations of the data (e.g., inspecting graphical representationssuch as bar or line graphs).

The process of comparing may be manual (such as visual inspection by thepractitioner of the method) or it may be automated. For example, anassay device (such as a luminometer for measuring chemiluminescentsignals) may include circuitry and software enabling it to compare ameasured value with a reference value for a biomarker protein.Alternately, a separate device (e.g., a digital computer) may be used tocompare the measured value(s) and the reference value(s). Automateddevices for comparison may include stored reference values for thebiomarker protein(s) being measured, or they may compare the measuredvalue(s) with reference values that are derived from contemporaneouslymeasured reference samples (e.g., samples from control subjects).

As will be apparent to those of skill in the art, when replicatemeasurements are taken, the measured value that is compared with thereference value is a value that takes into account the replicatemeasurements. The replicate measurements may be taken into account byusing either the mean or median of the measured values as the “measuredvalue.”

The invention also includes methods of identifying animals forparticular treatments or selecting animals for which a particulartreatment would be desirable or contraindicated.

The methods above may be performed by a reference laboratory, aveterinary hospital pathology laboratory, a university veterinarylaboratory, a veterinarian's office or a veterinarian. The methods abovemay further comprise an algorithm and/or statistical analysis.

5.5. Samples

The sample may be a urine sample, a tissue sample, a blood sample, acell free extract of blood, plasma, serum, urine. For the cytogeneticassays, as shown the examples, cells are used to provide templates forthe FISH probes. For PCR assays, tumor DNA may be obtained from cells orcell-free plasma/serum/urine.

5.6. Compositions and Kits

The invention provides compositions and kits for detecting urogenitalmalignancy in a dog comprising: (a) at least one reagent selected fromthe group consisting of: a nucleic acid probe capable of specificallydetecting CFA 13, CFA 19 or CFA 36; and (b) instructions for use inmeasuring a copy number of CFA 13, CFA 19 or CFA 36 in a biologicalsample from a dog wherein if the copy number of CFA 13 or CFA 36 iselevated, or CFA 19 is reduced, from that of a normal control.

The instructions comprise determining in a sample of relevant cellsobtained from the dog the presence of chromosomal abnormalities, whereinthe presence of chromosomal abnormalities involving at least two of theprobes indicates that the patient has bladder cancer. Such kits mayfurther comprise, or consist of, blocking agents or other probes,various labels or labeling agents to facilitate detection of the probes,reagents for hybridization (e.g., buffers), a metaphase spread, and thelike.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The article “a” and “an” areused herein to refer to one or more than one (i.e., to at least one) ofthe grammatical object(s) of the article. By way of example, “anelement” means one or more elements.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps. The present inventionmay suitably “comprise”, “consist of”, or “consist essentially of”, thesteps, elements, and/or reagents described in the claims.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely”,“only” and the like in connection with the recitation of claim elements,or the use of a “negative” limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The following Examples further illustrate the invention and are notintended to limit the scope of the invention. In particular, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

6. EXAMPLES

6.1. Experimental Data

Primary tumor biopsy samples (n=31) were collected from untreated dogswith TCC and either fixed in formalin or snap frozen prior to beingstored in liquid nitrogen. In addition non-neoplastic bladder tissueswere collected at North Carolina State University College of VeterinaryMedicine during necropsy of dogs with no signs of clinical disease.Control specimens were assessed by a veterinary pathologist andconfirmed to be histopathologically ‘normal’ with no signs of neoplasia.

6.2. oaCGH

DNA was extracted from tumors and verified to be high molecular weightupon agarose gel electrophoresis and with spectrophotometer readings of260:230>2 and 260:280>1.8 (Nanodrop-1000, Nanodrop). DNA isolated fromeach of the primary tumor biopsies (test samples) and labeled using theGenomic DNA Enzymatic Labeling Kit (Agilent) to incorporate afluorophore-conjugated dNTP, as described previously [2]. Genderspecific reference DNA samples were generated from mixed breed dogs,pooling equimolar quantities of DNA from 10 healthy males and 10 healthyfemales and labeled similarly but with a different fluorophore-conjugatedNTP. Fluorescently labeled test and reference samples were hybridizedto Canine G3 Sureprint 180,000 feature oligonucleotide-array-cCGH(oaCGH) arrays (Agilent, AMADID 025522) for 40 hours at 65° C. and 20rpm, as described previously [3]. Arrays were scanned at 3 μm using ahigh-resolution microarray scanner (Agilent, Model G2505C) and dataextracted using Feature Extraction (v10.9) software. Scan data wereassessed for data quality by the ‘Quality Metrics’ report in Agilent'sFeature extraction software (v10.5)(Agilent Technologies). FASST2Segmentation Algorithm, a Hidden Markov Model (HMM) based approach, wasused to make copy number calls. The FASST2 algorithm, unlike othercommon HMM methods for copy number estimation, does not aim to estimatethe copy number state at each probe but uses many states to cover morepossibilities, such as mosaic events. These state values are then usedto make calls based on a log-ratio threshold. The significance thresholdfor segmentation was set at 5×10⁻⁶, also requiring a minimum of threeprobes per segment and a maximum probe spacing of 1 Mb between adjacentprobes before breaking a segment. The log ratio thresholds for singlecopy gain and single copy loss were set at +0.201 and −0.234,respectively.

DNA copy number aberrations ‘called’ by the FASST2 segmentationalgorithm were compared between the cohorts of confirmed cases TCC(n=31) and clinical, healthy specimens (n>100). CGH results wereanalyzed using Nexus DNA Copy Number Discovery (V7). to identifyrecurrent aberrations in the cell population from which genomic DNA wasisolated; detectable aberrations represent the mean DNA copy number ofgenomic segments within that population of cells. Chromosomes with thehighest levels of recurrent DNA copy number changes in TCC identified byCGH were subsequently assessed for copy number status in individualcells obtained from urine specimens of canine patients presenting withconfirmed TCC as well as from dogs that were clinical health andpresented with no evidence of a malignancy.

6.3. Results

Genome-wide oaCGH data of DNA derived from biopsy specimens obtainedfrom 31 primary canine TCC cases revealed numerous copy numberaberrations across the canine genome, combinations of which could beused to develop an assay to detect the presence of aberrationsconsistent with the presentation of a TCC (FIG. 1).

FIG. 1. Frequency of significant DNA copy number changes in 31 cases ofcanine TCC. A) Genome-wide penetrance plot showing the distribution andfrequency of DNA copy number changes at (26 kb resolution). The x-axisshows the genome of the domestic dog partitioned into the 38 autosomesand the X chromosome. The y-axis shows the proportion of the 31 casesthat presented with a copy number aberration at each of the genomeintervals detected by the oaCGH platform used (smallest interval ˜26kb). Dark gray bars above 0% indicate the percentage of cases presentingwith DNA copy number increase and light gray bars below 0% indicate thepercentage of cases presenting with DNA copy number loss, at each of thesegmented regions. It is clear that there are numerous whole chromosomeand chromosomal segments that present with a level of copy numberaberration in the cohort evaluated. Any of these regions could be usedto detect the presence abnormal cells in patients suspected ofpresenting with a uriogenital malignancy. The highest frequencyaberrations (>75% of cases) involved DNA copy number increase of CFA 13and 36 and DNA copy number loss of CFA 19. A second tier of recurrentcopy number changes (where frequency is >33% of cases, represented bythe thicker black horizontal line) are evident as copy number loss ofregions of CFA 2, 5, 6, 10, 12, 26, 27, 28 and X, and copy numberinvolving regions of CFA 2, 4, 5, 6, 7, 10, 14, 17, 20, 23, 24, 30, 31,35, 38 and X. For the three most recurrent aberrations (gain of 13, lossof 19 and gain of 36), example peaks of aberration frequencyrepresenting ˜1 Mb intervals (detected by the specific oaCGH platformused) are shown by the vertical grey lines on CFA 13, 19 and 36. Inaddition the region of CFA 8 bisected by a grey vertical line representsthe region of the genome where no detectable significant copy changeswere evident in all 31 cases of TCC. FIG. 1B) Penetrance data presentedin a conventional cytogenetic ideogram of the canine karyotype toprovide an alternate representation of the chromosomes and chromosomalregions subject to DNA copy number gain (histograms to the right of achromosome) and loss (histograms to the left of each chromosome). FIG.1C) Individual chromosome penetrance plots showing the frequency of DNAcopy number changes along the lengths of CFA 8, 13, 19 and 38. The samegrey vertical lines indicated in panel A are retained for eachindividual chromosome to represent selected regions of each chromosomewhere the frequency of the aberration is highest, for CFA 13, 19 and 38,and where no copy number changes were detected in any of the 31 cases onCFA 8. The example 1 Mb sized peaks of aberration frequency (indicatedby FASST2 analysis of log 2 ratios) indicated on CFA 13, 19 and 36 arecentered at chr13:35-36 Mb, chr19:25-26 Mb and chr36:23-24 Mb, and theregion of CFA8 that was copy number neutral when assessed by FASST2analysis of log 2 ratio data was located at chr8:7-8 Mb using canfam2locations.

For the three chromosomes with the highest frequency of aneuploidy,represented by gain of dog chromosome (CFA) 13, deletion of CFA 19, andgain of CFA 36, the frequencies of these changes in the samplesevaluated are shown in Table 1. Analysis of over 100 cases ofnon-neoplastic specimens from dogs indicated no detectable copy numberchanges of each of CFA 13, CFA 19 and CFA 36 for this sample set. Thesedata therefore provide a means to determine the presence of abnormalcells based on detection and enumeration of one or more of these threechromosomes.

TABLE 1 Frequency of categorical (neutral, loss or gain) copy numberstatus of dog chromosomes 13, 19 and 31 in cases and controls,determined by analysis of DNA samples isolated form biopsy specimens of31 cases of confirmed canine TCC and >100 cases of non-malignant cells.The expected copy number of all three of these chromosomes is n = 2(neutral), as evident by analysis of cells from healthy dogs. The copynumber changes detected for CFA 13, 19 and 36 were unidirectional, withCFA 13 and 36 present as additional copies and CFA 19 presented withreduced copies. TCC ‘cases’ (n = 31) Frequency of copy number statusChromosome gain (n > 2) loss (n < 2) neutral (n = 2) CFA 13 96.77% 0.00%3.23% CFA 19 0.00% 77.42% 22.58% CFA 36 83.87% 0.00% 16.13%

Normal ‘controls’ (n > 100) Chromosome gain (n > 2) loss (n < 2) neutral(n = 2) CFA 13 0.00% 0.00% 100.00% CFA 19 0.00% 0.00% 100.00% CFA 360.00% 0.00% 100.00%Table 2 shows additional data regarding the 31 samples.

TABLE 2 Combined frequencies of copy number gain of 36, and 19 and lossof 19 in 31 cases of canine TCC. Aberration in each case Numberproportion % age +13 and −19 and +36 21 21/31  67.74% +13 and −19 only 22/31 6.45% +13 and +36 only 5 5/31 16.13% −19 and +36 only 1 1/31 3.23%+13 only 2 2/31 6.45% TOTAL 31 100.00%these data show that

-   100% of TCC have one or more of the three aberrations-   93.55% of TCC have two or more of the three aberrations-   67.74% of TCC have all three of the aberrations

6.4. Statistical analysis

Analysis of several hundred DNA samples from pathologically verifiednon-neoplastic tissue (controls) indicated that none of CFA 13, CFA 19and CFA 36 demonstrated copy number aberration. As such the sensitivity,specificity, % correctly classified, and the AUC values (with 95% CI)for each of the three regions were calculated, based on the presence ofat least 100 totally negative controls.

Using the frequencies provided in Table 1 for the aneuploidy of dogchromosomes 13, 19 and 36 in canine TCC, measures of association andpotential predictive performance were calculated for each of threeaberrations.

Several statistical measures were calculated.

First, the relative risk (RR) was calculated. As calculated, the riskratio can be interpreted as the overall risk of a dog presenting withneoplastic cells detectable (in either a bladder tumor biopsy or a urinesample from a suspected cases of TCC) given that it has gain of CFA 13and/or gain of CFA 36 and/or loss of CFA 19, compared to the overallrisk that a dog does not have neoplastic cells. Relative risk (RR) issimply the probability or relationship between two events. For example,a relative risk of 10 would indicate that patient from which thespecimen was obtained would be 10 times more likely to have a urinarytract neoplasm/TCC than not.

Second, the odds ratio (OR) was calculated. As calculated, the oddsratio can be interpreted as the odds of a dog presenting with amalignancy/TCC, given that it has aneuploidy of the chromosomeevaluated, compared to the odds that a dog does not have amalignancy/TCC given that it has aneuploidy of the chromosome evaluated.Instead of using pure percentages (as for RR), OR uses the ratio ofodds. The OR explains the ‘odds’ not in its colloquial definition (i.e.chance) but rather on its statistical definition, which is theprobability of an event over (divided by) the probability of a certainevent not happening.

Third, the sensitivity and specificity were calculated to determinelikelihood of false positives and false negatives. Sensitivity measuresthe proportion of actual positives that are correctly identified as such(in this case the percentage of confirmed cases of TCC correctlyidentified). Specificity measures the proportion of negatives that arecorrectly identified, in this case the percentage of confirmed non-TCCbearing dogs correctly identified.

Fourth, an overall misclassification rate was calculated. This measuretells the percentage of dogs that are misclassified by this marker. Theaccuracy of the test overall would simply be one minus (1−) themisclassification rate.

Additionally, 95% confidence intervals were calculated for each of thesemeasures for each region.

The statistical findings and their interpretations are listed below foreach of the three regions individually.

TABLE 3 Segment 1: Gain of chromosome 13 95% Confidence Measure ValueInterval Relative Risk 101.00 23.044 101.00 Odds Ratio Infinite NA NASensitivity 0.968 0.873 0.968 Specificity 1.00 0.970 1.00Misclassification Rate 0.008 0.008 0.053

TABLE 4 Segment 2: Loss of chromosome 19 95% Confidence Measure ValueInterval Relative Risk 15.286 8.786 9.417 Odds Ratio Infinite NA NASensitivity 0.774 0.663 0.774 Specificity 1.00 0.966 1.00 Misclassification Rate 0.053 0.053 0.106

TABLE 5 Segment 3: Gain of chromosome 36 95% Confidence Measure ValueInterval Relative Risk 21.00 10.953 21.00 Odds Ratio Infinite NA NASensitivity 0.839 0.731 0.839 Specificity 1.00 0.966 1.00Misclassification Rate 0.038 0.038 0.089

In all three cases, since aneuploidy of chromosomes 13, 19 and 36 werenot detected in specimens from any of >100 ‘healthy’ dogs, aneuploidyfor each of the regions provides a high level of specificity (>99% inthe current data set) that the dog from which the biopsy/urine samplewas taken presented with neoplastic cells/TCC. Further, the physiologyof the dog would suggest that any aberrant cells are highly likely to bederived from the bladder or urogenital tract and so the chances of theaberrant cancer not being of urogenital origin is very small. Inconsideration of the data presented, we may deduce that the combinationof these aberrations is consistent with that expected from cells derivedfrom a canine TCC.

6.5. Combinatorial Analysis.

To evaluate the potential predictive power of a multivariate model(using up to all three regions together, with gain and loss informationfor all three regions included), a decision tree model was constructedusing the J48 algorithm. With or without cross-validation, the best treeonly had a single variable included, copy number gain of CFA 13. Thiswas the best model, adding the additional variables neither improved norweakened the model.

In addition, if cells from a urinary tract present with two or more ofthe three aberrations above, the sensitivity and specify to indicate thepresence of neoplasm is extremely high (>99%, based on the data setevaluated).

The detection of the two most frequent aneuploidies, gain of CFA 13/gainof CFA 36, renders an OR of 422.230, a RR of 33.817, andmisclassification is 0. All confidence intervals are undefined.

The data described above provide the basis for the discovery that;

the detection of one or more of three major aberrations, defined as copynumber gain of CFA 13, copy number loss of CFA 19, and copy number gainof CFA 36, whether in whole or part, in cellular specimens obtained fromthe urogenital tract of a dog, male or female, whether the specimenrepresents a biopsy of the suspect mass, or urine, would indicate thepresence of abnormal cells, where such cells could be described asneoplastic and also as likely derived from a transitional cell carcinomaof the urogenital tract.

Any method that is able to detect and quantify the copy number status ofCFA 13 and/or, CFA 19 and/or CFA 36, either in part or in entirety, maybe used to enable this invention. Such approaches may include, but arenot restricted to,

(1) conventional cytogenetic assessment of metaphase preparationsobtained from canine patient samples,

(2) fluorescence in situ hybridization (FISH) whereby one or more probesrepresenting part or all of CFA 13 and/or 19 and/or 36 are brought intocontact with cells derived from canine patients, be they cells from amass or from a urine sample of the patient. Such probes may, though arenot restricted to, those representing the full or partial length of thechromosomes being detected and quantified (e.g., whole or partialchromosome paint probes), probes classified as ‘locus specific’ andwhich bind to a specific regions of the chromosome being assessed, andwhich have been determined suitable for the detection and quantificationof that region. Single locus probes may comprise one or more fragmentsof single stranded or double stranded nucleic acid, in some forms ascloned fragments propagated in a bacterial of bacteriophage host (e.g.,BAC, PAC, phage, cosmid, plasmid and others) and in other formsgenerated by PCR amplification of the specific sequences reported in thegenome sequence. In these case the probes may be labeled with a haptenand subject to detection with a suitable chromogen or fluorophore posthybridization, using procedures widely known to those skilled in theart, or may themselves be labeled with a fluorophore prior to thehybridization so that they may report the site of hybridizationimmediately following hybridization and suitable washing steps knownwidely to those skilled in the art. In an alternate form the probe maycomprise collections of numerous single or double stranded nucleic acidsequences (e.g. oligonucleotides that may be of varying length), wherethe sequences are designed based on available nucleic acid sequencesrepresenting the regions to be detected and enumerated, which may belabeled with a fluorophore. For example, but not limited to, such probesmay be designed to detect the regions defined by the grey vertical linesin FIG. 1, representing the peak of the aberration frequencies in thecohort of TCC cases assessed, recognizing that any of these preciselocations may move as additional cases are evaluated.

(3) polymerase chain reaction (PCR) using DNA isolated from patientsamples; amplification based methods are used widely to obtainedrelative and absolute copy number of nuclei acid in a specimen, be that,for example, conventional PCR, quantitative PCR (qPCR) or dropletdigital PCR (ddPCR). For example, in the design of a qPCR or ddPCRassay, the determination of the absolute concentration of ampliconsbased on the TCC samples evaluated would require the amplification ofsequences that reside on CFA 13, 19 and 36, optionally in the regionsrepresenting the peaks of each aberration. For example, the verticalgrey lines in FIGS. 1A and 1C represent selected ˜1 Mb intervalsdemonstrating example peaks of aberration for CFA 13, 19 and 36(indicated by FASST2 analysis of log 2 rations) and a region of CFA 8that was copy number neutral when assessed by FASST2 analysis of log 2ratio data. Each of the above base locations are derived from theboundaries of copy number events and derived from canfam2 (UCSC genomebrowser) and could be relocated into the current version of caninegenome assembly (CanFam3), publicly available at ENSEMBLE and USCS. Useof a nucleic sequence within, but not restricted to this small region ofCFA 8 may thus be used to establish a neutral copy number status in TCCwhen using qPCR and ddPCR, from which the relative concentration ofamplicons (and thus copy number) may be deduced for regions assessed forcopy number aberration, in this example, but not limited to the definedbasepair locations noted on CFA 13, 19 and 36.

(4) comparative genomic hybridization. Any format of array that canprovide an indication of copy number of a ‘test’ specimen may be used.Many methods for using immobilized nucleic acids on a range of solidsurfaces are widely known to those skilled in the art.

(5) Next generation sequencing methods can determine that the abundanceof the target regions as being greater than or less than expected basedon the abundance of regions known to be normal.

Example 1

To provide an example of how one of the above approaches may be used inpractice, a four color cytogenetic assay was developed using caninebacterial artificial chromosome (BAC) clones designed to hybridize toeach of these chromosomes. Canine BAC clones (Table 5) were selectedfrom the CHORI-82 canine library to provide a means to detect andquantify dog chromosomes 13, 19 and 36 by fluorescence in situhybridization (FISH) of cells obtained from canines. A fourth FISH probewas developed to represent a region of dog chromosome 8 that wasobserved to be copy number neutral in all 31 cases of TCC biopsiesevaluated.

TABLE 6 Identification of four canine BAC clones from the CHORI-82canine BAC library that were used to provide a means to detect andquantify the copy number of CFA 8, CFA 13, CFA 19 and CFA 36 when usedin multicolor FISH analysis of individual cells obtained from patientsamples with confirmed TCC. All four BACs are selected from the setcytogenetically verified in house and reported previously [4].Chromosome Probe Target CHORI-82 location size Label chromosome clone(bp) (canfam2) (bp) color CFA 8 CH82-122f23 7984903-8129058 144156 goldCFA 13 CH82-328p06 8630922-8818498 187577 red CFA 19 CH82-122m0125119452-25264995 145543 green CFA 36 CH82-122f03 23199386-2326976670380 Pink (cy5)

Four color FISH was performed on cells isolated from free-catch urinesamples obtained from healthy dogs and from dogs with a confirmeddiagnosis of TCC. DNA was prepared from the BAC clones identified inTable 6 and labeled to incorporate one of four spectrally resolvablefluorochrome conjugate dNTPs, each using standard protocols and which wehave published previously e.g. [4, 5]. The four probes were mixed andhybridized to cells isolated from urine obtained from healthy and cancerbearing dogs, using protocols used widely in the art and which we havepublished previously e.g. [4, 5]. The inclusion of unlabeled dog DNArepresenting repetitive elements dispersed throughout the genome wasincluded to suppress hybridization of any repetitive element within thefour BAC probes to genomic sites other than their primary uniquelocation in the canine genome. Images were acquired using a multicolorFISH workstation equipment with narrow pass fluorescence filters forwavelengths representing light seen in color as gold, red, green and Cy5(far red) and a cooled CCD camera. Cell nuclei were counterstained inDAPI. Each color plane was imaged as black and white and then thefluorescence signal detected for each plane was pseudo-colored torecapitulate the color associated with the wavelengths passing througheach filter (Cy5 signal is far red and so was presented as a pink)

FIG. 2 illustrates the hybridization characteristics of all four probeswhen contacted with the chromosome of a non-neoplastic cell,demonstrating that, under the conditions used (standard for those in theart), each of the four probes detects two copies of the specific andunique region of the canine genome, localized at the designated physicallocation of the appropriate pair of homologous chromosomes.

FIG. 2. Multicolor Hybridization of Fluorescence In Situ HybridizationProbes Designed to Detect and Quantify Dog Chromosomes 8, 13, 19 and 36.

In cases of canine urogenital carcinoma/TCC, dog chromosome (CFA) 13 and36 are subject to copy number increase (n>2), while CFA 19 is subject tocopy number decrease (n<2). In the same cells CFA 8 has a balanced copynumber (n=2). In this example canine BAC clones were selected torepresent CFA 8, 13, 19 and 36 at the Mb locations in panel C. DNA fromthe four BAC clones was labeled for use in fluorescence in situhybridization (FISH) analysis using the incorporation of four spectrallyresolvable fluorophores enabling detection as separate sites ofhybridization; CFA8=gold, CFA13=red, CFA 19=green, CFA 36=pink (shown asgray scale). FIG. 2A shows the four FISH probes hybridized to a dogmetaphase preparation from a cell obtained from healthy dog, where thedog chromosomes are counterstained in DAPI, resulting in a blue color.FIG. 2B is the same image as panel A, processed to reveal the DAPIbanding in gray scale. FIGS. 2A and 2B demonstrate that each probehybridizes to just two regions of the genome, with each siterepresenting the homologues of chromosomes 8, 13, 19 and 36. FIG. 2Cshows one of each pair of homologues from panel B now enlarged andcorrectly oriented to demonstrate the chromosomal location of thehybridization signal. Beside each chromosome is shown the chromosomenumber and the precise physical location on that chromosome (in Mb) towhich the selected probe binds.

FIG. 3 illustrates the typical pattern of hybridization of the same fourprobes when hybridized to a non-neoplastic cell from a urine specimen ofa healthy dog and to cells obtained from free catch urine obtained fromdogs with confirmed transitional cell carcinoma. In healthy cells thecopy number of all four probes is n=2. In the two cells shown torepresent examples of those obtained from TCC bearing dogs, while thecopy number of the probe representing CFA 8 is n=2, the copy number ofthe probe representing CFA 19 is n=1 and the copy number of the probesrepresenting CFA 13 and CFA 16 are both n>2.

FIG. 3. Examples of using multicolor (five-plane) FISH analysis todetect and quantify probes representing CFA 8 (gold), 13 (red), 19(green) and 36 (pink) in DAPI (blue counterstain) (shown as gray scale)cells obtained from urine samples of A) a healthy dog and B&C) two dogswith a confirmed diagnosis of a transitional cell carcinoma of thebladder. In Ai/Bi/Ci the five-color plane image is shown and inAii/Bii/Cii the same five-color plane is presented with the sites ofhybridization each arrowed with the corresponding color.

In Panel A/Ai it is evident that all four probes are present as twodistinct copies (n=2), indicative of a ‘healthy/copy number balanced’status. In B/Bi and C/Ci both cells have a balanced copy number of theCFA 8 probe (n=2), as expected, while each has just one copy of theprobe representing CFA 8 (green) and multiple copies of the probesrepresenting CFA 13 (red) and CFA 36 (pink) (shown as gray scale). Thelarge size of some of the hybridization sites for the probesrepresenting CFA 13 (red) and CFA 36 (pink) is indicative of tandemduplications at the sites of hybridization and so determination of theprecise copy number per cell, over the actual number of visible sites ofhybridization, is not possible. We may therefore state that in cells Band C the copy number of the probes representing CFA 13 are n>5 and n>4,respectively, and that in cells B and C the copy number of the proberepresenting CFA 36 is n>7 and n>6, respectively. In these two cellsboth have aneuploidy of all three target regions.

The example shown above may be used to represent a ‘kit’ suitable fordetecting and evaluating the target chromosomes in canine cells. Toevaluate the frequency of aneuploidy of the three target chromosomeswithin cells shed from the urogenital tract of dogs diagnosed with TCC,we evaluated up to 30 cells in each of 10 urine samples. Across all 10cases the frequency of cells with aberrant copy mean number of cells foreach of the three target regions and the number range in frequencies ofaberration are show in Table 6. These data demonstrate that among thecases evaluated the minimum number of cells aberrant for at least one ofthe three regions was 23%.

TABLE 7 Summary of proportions of cells exhibiting copy numberaberrations of CFA 13, CFA 19 and CFA 36 in populations of cellsobtained from urine samples of dogs with a confirmed diagnosis of TCC.Frequency of aberrant Mean % of aberrant cells Aberration cells per caseper case +CFA 13 55%-100% 88% −CFA 19 23%-80%  41% +CFA 36 35%-100% 84%

Further, in eight of the ten cases evaluated, 100% of the cells had oneor more of the three target aberrations, while the remaining two caseshad 93% and 87% of cells with one or more of the target aberrations.These data provide sufficient evidence to allow the implementation of adiagnostic FISH based assay to confirm the presence of abnormal cells,highly suggestive of a TCC, by detection and quantification of the threechromosomes above, based on enumeration of cells shed from theurogenital tract into the urine.

Example 2

To provide an example of how this invention may be used in the form of aquantitative PCR assay, the invention was also reduced to practice inthe form of a droplet digital PCR (ddPCR) assay. The peaks of copynumber aberration for CFA 13, 19 and 36 were defined as shown by thegrey vertical lines in FIG. 1; representing a ˜1 Mb regions of CFA 13 atchr13:35-36 Mb, CFA 19 at chr19:25-26 Mb and CFA 36 at chr36:23-24 Mbusing canfam2. In addition, the copy number neutral region of CFA8,located at chr8:7-8 Mb was included to represent a region of the genomeunchanged in copy number in canine TCC.

The DNA sequence of the canine genome within each of the three definedregions of aberration on CFA 13, 19 and 36, and the ‘neutral’ region onCFA 8, were evaluated and used to design four TaqMan® MGB assayssuitable for use in DNA copy number analysis The details of each of thefour assays are presented in Table 8. In this example the BioRad QX100droplet digital PCR system was used to determine copy number of thethree target assays (located on CFA 13, 19 and 36) relative to that ofthe ‘copy number neutral assay located on CFA 8.

Table 8. DNA sequences (from canfam2) and genome locations of each PCRprimer and TaqMan probes used in the four paired assays developed todetect and quantify copy number of specific regions of CFA 13, 19 and 36in cases of canine TCC. For each copy number assay one of the three testassays is mixed with the reference assay and the PCR run as a duplexreaction. The assay shows were developed to work with equal performanceusing the same PCR cycling conditions. Probes, primer and amplicons areSEQ ID NOS:1-16.

TABLE 8 TCC ddPCR assays label primer/probe sequence (5′-3′) start baseend base Reference assay CFA 8 n/a forward primer CCAGGATTCTGCAGAGTTTGATCFA8:7695383 CFA8:7695404 SEQ ID NO: 1 HEX TaqMan probeAATGCCTTTGACCAGTGGGTAGCC CFA8:7695347 CFA8:7695370 SEQ ID NO: 2 n/areverse primer GTGGTGGAGGATTTGGAAGAAG CFA8:7695302 CFA8:7695323SEQ ID NO: 3 amplicon Ccaggattctgcagagtttgatttgttgtttgaaaatgcc amplicon103 bp sequence tttgaccagtgggtagccagcacagcctcagaaaaatgca lengthccttcttccaaatcctccaccac SEQ ID NO: 4 Test assay CFA 13 n/aforward primer AGGACTATGTGTAAATCAGTAAGATAGG CFA13:35393771CFA13:35393798 SEQ ID NO: 5 FAM TaqMan probe TGAATGAGGTTGAGGATGAAGCAAGGTCFA13:35393822  CFA13:35393848 SEQ ID NO: 6 n/a reverse primerCCCACTTCATGACCATATCCC CFA13:35393854 CFA13:35393874 SEQ ID NO: 7amplicon Aggactatgtgtaaatcagtaagataggcctgtatagtatgaaga amplicon 104 bpsequence atggattgaatgaggttgaggatgaagcaaggttagttgggatat lengthggtcatgaagtggg SEQ ID NO: 8 CFA 19 n/a forward primerGGACTGCTGAACTTCCTTCAT CFA19:25520681 CFA19:25520701 SEQ ID NO: 9 FAMTaqMan probe AGGTCCAATATTGCAATGAGTGAAGCA CFA19:25520739 CFA19:25520765SEQ ID NO: 10 n/a reverse primer CTGTGGCTTCCTCATCGTTT CFA19:25520768CFA19:25520787 SEQ ID NO: 11 ampliconggactgctgaacttccttcatttctcagtcatcagcaaactaaga amplicon 107 bp sequencegaagtaaactttaagGtccaatattgcaatgagtgaagcataaaa length cgatgaggaagccacagSEQ ID NO: 12 CFA 36 n/a forward primer TGCATGTCAAGAGAGGAGAATGCFA36:23654179 CFA36:23654200 SEQ ID NO: 13 FAM TaqMan probeCCACTTGCTCATATGTACACCTGATTGTCC CFA36:23654229 CFA36:23654258SEQ ID NO: 14 n/a reverse primer TCCAGCTTGCAGAGTTGTT CFA36:23654260CFA36:23654279 SEQ ID NO: 15 ampliconTgcatgtcaagagaggagaatgtgagaattttaattgctctaaataa amplicon 101 bp sequenceagaccacttgctcatatgtacacctgattgtccaaaacaactctgca length agctggaSEQ ID NO: 16

Each TaqMan assay was designed to provide an amplicon of 100-110 bp,with each amplified using the same thermal cycling conditions. DNA wasisolated from the same urine samples used to provide material for FISHanalysis shown as example 1, so that data generated by FISH and ddPCRwere of the same cases. The ‘reference’ assay (CFA 8) was labeled withHEX and the three ‘test’ assays (CFA 13, 19 and 36) were each labeledwith FAM. Each copy number assessment was processed as a pair of TaqManassays in a single tube/well, comprising the reference assay and one ofthe three test assays. For each duplex/paired reaction, reactioncomponents (i.e. sample DNA, PCR master mix, forward and reverseprimers, HEX labeled TaqMan probe for the reference region on CFA 8 andFAM labeled TaqMan probe for the test region on either of CFA 13 or 19or 36), were mixed into a single tube/well. The QX100 droplet generatorwas the used to partition each sample into up to 20,000 onenanoliter-sized droplets. Following thermal cycling of the reagentswithin each droplet, using protocols known well to those in the art,droplets from each sample were streamed in single file in the QX100droplet reader and the number of positive and negative droplets for eachof the test and reference amplicons was detected. Theamplicon/PCR-positive and amplicon/PCR-negative droplets were counted toprovide absolute quantification in digital form. When theconcentration/microliter of each of the three test assays werenormalized to the concentration/microliter of the reference assay in thesame tube/well, the a mean copy number of the ‘test’ region in the DNAsample was determined. Data for all three paired assays are shown inFIG. 4, indicating that ddPCR is able to accurately detect and quantifythe copy number status of aberrant regions, providing an alternate meansto FISH based assessment for identification of aberrant DNA samples.

FIG. 4. Example of the use of ddPCR to determine the mean DNA copynumber of aberrant regions of the canine genome in DNA samples obtainedfrom urine specimens of TCC bearing dogs. The inset FISH image is thesame cell as shown in FIG. 3, panel B, where the copy number of thesingle locus probes for CFA 8, 13, 19 and 36 were recorded as n=2, n=>5,n=1 and n=>7. When 30 cells from that TCC patient were analyzed, themean copy number of each of the probes remained constant. ddPCR data forthe three aberrant regions are shown in the main body of the figure. Thechart for each region is presented with three data points, theconcentration of the target sequence/amplicon (i.e., of CFA 13 or 19 or36), the concentration of the reference sequence/amplicon (i.e. CFA 8),and the derived mean copy number of the target sequence/amplicon(arrowed) obtained by normalization to the concentration of thereference sequence/amplicon (CFA 8). A summary of the mean copy numberfor each of the four regions (CFA 8, 13, 19 and 36), by assessment of 30individual cells using the FISH approach reported in example 1, ispresented in the table. The mean DNA copy number for the ‘test’ regions(CFA 13, 19 and 36) relative to the ‘reference region (CFA 8). Resultingfrom an assessment of 16,000 1 nl droplet PCR reactions, is also shown.

The ddPCR derived mean copy number of the regions on CFA 13, CFA 19 andCFA 36 were n=6.19, n=1.07 and n=27.1. The value for the mean copynumber of the region on CFA 19 (n=1.07) is comparable to the mean valueobtained by analysis of 30 individual cells (n=1.02). For CFA 13 and CFA36, the FISH data indicated that the mean copy number was n>5 and n>7,respectively, while the ddPCR data for these two regions indicate meancopy numbers of n=6.19 and n=27.1, respectively. These data support thebasis of using a ddPCR based assay to accurately detect and quantifycopy number of target regions, providing added value to FISH analysisfor enumeration of regions where the additional copies are tandemlyduplicated and thus non-resolvable by FISH.

7. REFERENCES

-   1. Mutsaers, A. J., W. R. Widmer, and D. W. Knapp, Canine    transitional cell carcinoma. J Vet Intern Med, 2003. 17(2): p.    136-44.-   2. Thomas, R., et al., Refining tumor-associated aneuploidy through    ‘genomic recoding’ of recurrent DNA copy number aberrations in 150    canine non-Hodgkin lymphomas. Leukemia & lymphoma, 2011. 52(7): p.    1321-35.-   3. Seiser, E. L., et al., Reading between the lines: molecular    characterization of five widely used canine lymphoid tumour cell    lines. Veterinary and comparative oncology, 2011.-   4. Thomas, R., et al., A genome assembly-integrated dog 1 Mb BAC    microarray: a cytogenetic resource for canine cancer studies and    comparative genomic analysis. Cytogenet Genome Res, 2008. 122(2): p.    110-21.-   5. Breen, M., et al., An integrated 4249 marker FISH/RH map of the    canine genome. BMC Genomics, 2004. 5(1): p. 65.

It is to be understood that, while the invention has been described inconjunction with the detailed description, thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below. All publications,patents, and patent applications cited in this specification are hereinincorporated by reference as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method for diagnosing and treating a urogenitalmalignancy in a dog, the method comprising: (a) measuring a copy numberof CFA 13, CFA 19, CFA 36, or any combination thereof in a biologicalsample from a dog; wherein: (i) the biological sample is selected fromthe group consisting of a urine sample, cells isolated from the urinarytract of the dog, and a combination thereof; and (ii) the copy number ofCFA 13, 19, and/or 36 is measured by aberrations at CFA chr13:35-36 Mb,CFA chr13:8630922-8818498, CFA chr19:25-26 Mb, and/or CFA chr36:23-24 Mbusing canfam2; (b) detecting an elevated copy number of CFA 13 and/orCFA 36 compared to a normal control or a reduced copy number of CFA 19compared to a normal control; (c) diagnosing a urogenital malignancy inthe dog; and (d) treating the urogenital malignancy in the dog withsurgery, radiation therapy, chemotherapy, or any combination thereof. 2.The method of claim 1, wherein the copy number of CFA 13, CFA 19 and CFA36 are measured.
 3. The method of claim 1, wherein the copy number(s) ismeasured by fluorescence in situ hybridization (FISH).
 4. The method ofclaim 1, wherein the copy number(s) is measured by polymerase chainreaction (PCR).
 5. The method of claim 1, wherein the copy number(s) ismeasured by comparative genomic hybridization (CCH).
 6. The method ofclaim 1, wherein the copy number(s) is measured by next generationsequencing.
 7. The method of claim 1, wherein the biological sample is aurine sample.
 8. The method of claim 1, wherein the sample is afresh-frozen sample.
 9. The method of claim 1, wherein the sample is afresh sample.
 10. The method of claim 1, wherein the sample is aformalin-fixed, paraffin-embedded sample.
 11. The method of claim 1,wherein aberrations at CFA chr13:8630922-8818498, CFAchr19:25119452-25264995, and CFA chr36:23199386-23269766 are measured.12. A method for selecting and treating a dog for a urogenitalmalignancy, the method comprising: (a) measuring a copy number of CFAchr13:35-36 Mb, CFA chr13:8630922-8818498, CFA chr19:25-26 Mb, and/orCFA chr36:23-24 Mb in a biological sample from a dog, wherein thebiological sample is selected from the group consisting of a urinesample, cells isolated from the urinary tract of the dog, or acombination thereof isolated from the dog using canfam2; (b) detectingan elevated copy number of CFA chr13:35-36 Mb, CFAchr13:8630922-8818498, and/or CFA chr36:23-24 Mb compared to a normalcontrol or a reduced copy number of CFA chr19:25-26 Mb compared to anormal control; (c) diagnosing a urogenital malignancy in the dog; and(d) selecting and treating the dog for the urogenital malignancy,wherein the treating comprises surgery, radiation therapy, chemotherapy,or any combination thereof.
 13. The method of claim 12, wherein the copynumbers of CFA chr13:35-36 Mb, CFA chr19:25-26 Mb, and CFA chr36:23-24Mb are measured.
 14. The method of claim 12, wherein the sample is afresh-frozen sample.
 15. The method of claim 12, wherein the sample is afresh sample.
 16. The method of claim 12, wherein the sample is aformalin-fixed, paraffin-embedded sample.
 17. The method of claim 12,wherein aberrations at CFA chr13:8630922-8818498, CFAchr19:25119452-25264995, and CFA chr36:23199386-23269766 are measured.